Active site of human gamma glutamy hydrolase

ABSTRACT

Provided is the active site of human gamma glutamyl hydrolase. The active site resides in amino acid residues 110, 171, 220 and 222 of SEQ ID NO:1. Thus provided is an inactive gamma glutamyl hydrolase protein, as well as a fragment thereof. A method of inactivating a gamma glutamyl hydrolase protein is also provided, as is a molecule capable of binding to one or more of amino acid residues 110, 171, 220 or 222 of SEQ ID NO:1 which can be used in such a method. A method for identifying a molecule that inactivates gamma glutamyl hydrolase is provided, as is a nucleic acid molecule encoding the inactive gamma glutamyl hydrolase.

This application is a divisional of U.S. Ser. No. 09/326,157, filed Jun.4, 1999 now U.S. Pat. No. 6,573,077, which is continuation-in-part ofU.S. Ser. No. 09/128,722, filed Aug. 4, 1998, now U.S. Pat. No.5,962,235, which was a divisional of U.S. Ser. No. 08/628,291, filedApr. 5, 1996, now U.S. Pat. No. 5,801,031.

This invention was made with support from the United States Governmentunder Grant No. CA 25933 of the National Cancer Institute, NationalInstitutes of Health. The U.S. Government may have certain rights inthis invention.

FIELD OF THE INVENTION

The subject invention is directed generally to gamma glutamyl hydrolase,and more particularly to the identification of the active site withingamma glutamyl hydrolase.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced, many inparenthesis. Full citations for each of these publications are providedat the end of the Detailed Description. The disclosures of each of thesepublications in their entireties are hereby incorporated by reference inthis application.

Many enzymes involved in producing precursors for DNA synthesis requirefolate as a cofactor. Antifolate drugs which impair folate function,such as methotrexate (MTX), are the primary treatments for many cancers.The retention and efficacy of folates and antifolate drugs within thecell are dependent on the addition of a poly-γ-glutamate chain to themonoglutamate (McBurney and Whitmore 1974). Folylpolyglutamatesynthetase (FPGS) catalyzes the sequential addition of glutamate (forreviews see McGuire and Coward 1984; Shane 1989) and γ-glutamylhydrolase (GH, EC 3.4.19.9) catalyzes the removal of glutamate fromfolyl- and antifolylpoly-γ-glutamates (for review see Galivan and Ryan1998). In conjunction with the folate transport systems, the balancebetween GH and FPGS activity regulates the amount of glutamylation offolate and antifolate drugs in the cell.

GH activity in mammalian cells is found in the lysosomes (McGuire andCoward 1984; Hoffbrand and Peters 1969; Silink and Rowe 1975a; Wang etal. 1986; Yao et al. 1995). However, in cell culture, the major part ofthe synthesized enzyme is secreted into the medium (O'Connor et al.1991). In humans, GH activity has been detected in plasma (Baggott etal. 1987), bile (Horne et al. 1981), pancreatic juice (Bhandari et al.1990), and jejunal mucosa (Reisenauer et al. 1977). The enzyme has beenpurified from a number of mammalian tissues (Saini and Rosenberg 1974;Silink et al. 1975b; Rao and Norohna 1977; Elsenhans et al. 1984; Wanget al. 1993) and cell lines (Yao et al. 1995; O'Connor et al. 1991; Wanget al. 1993; Rhee et al. 1998). Both rat GH and human GH areglycoproteins (Rhee et al. 1998; Yao et al. 1996a). GH from differentspecies has a different specificity in the hydrolysis of thepoly-γ-glutamyl tail. For example, the rat GH enzyme acts as anendopeptidase (Wang et al. 1993) hydrolyzing the innermost γ-glutamylbond and releasing the poly-γ-glutamate chain as a single unit.Conversely GH isolated from human sources (hGH) removes only thecarboxyl terminal glutamate or di-γ-glutamate during the reaction (Rheeet al. 1998).

The cDNA's encoding GH from rat and human sources have been isolated(Yao et al. 1996a; Yao et al. 1996b; U.S. Pat. No. 5,801,031, issuedSep. 1, 1998 and incorporated herein by reference) and a mouse GH cDNAhas recently been isolated (Esaki et al. 1998). The hGH cDNA has beenexpressed in both an insect expression system (Rhee et al. 1998) andEscherichia coli (Yao et al. 1996b). The first 24 amino acids encoded bythe hGH cDNA are a signal peptide, which is removed during processing(Rhee et al. 1998). Therefore, the N-terminal amino acid of the maturehGH enzyme is equivalent to R25 in the published hGH sequence (Yao etal. 1996b).

Early studies demonstrated that GH is sulfhydryl sensitive and isinhibited by iodoacetic acid or p-hydroxymercuribenzoate (pHMB) (McGuireand Coward 1984; Reisenauer et al. 1977; Silink et al. 1975b). Studieson GH in lysosomes isolated from murine S180 cells indicate thataccumulation of reduced sulfhydryls in the lysosome activate GH(Barrueco et al. 1992). Recent studies with pure GH preparationsverified the earlier findings of sulfhydryl sensitivity (O'Connor et al.1991; Rhee et al. 1998; Yao et al. 1996a).

The catalytic mechanism of GH has yet to be defined. A betterunderstanding of the mechanism could lead to the specific inhibition ofthis enzyme and increased efficacy of antifolate drugs.

SUMMARY OF THE INVENTION

The subject invention addresses this need by identifying the active siteof GH as including amino acid residues 110, 171, 220 and 222 of SEQ IDNO:1. SEQ ID NO:1 represents the amino acid sequence of mature humannative GH (without the signal peptide). The subject invention thusprovides an inactive gamma glutamyl hydrolase protein, the inactiveprotein having an amino acid sequence that substantially corresponds tothe amino acid sequence of native gamma glutamyl hydrolase as shown inSEQ ID NO:1, SEQ ID NO:1 being modified at one or more of amino acidresidues 110, 171, 220 or 222 to render the resulting gamma glutamylhydrolase protein inactive. The invention further provides a fragment ofthe inactive gamma glutamyl hydrolase protein, wherein the fragment isfrom about 10 to about 150 amino acids in length and wherein thefragment includes one or more of the modified amino acid residues.

Also provided by the subject invention is a method of inactivating agamma glutamyl hydrolase protein. The method comprises: providing agamma glutamyl hydrolase protein; and modifying one or more of aminoacid residues 110, 171, 220 or 222 in the amino acid sequence of thegamma glutamyl hydrolase protein as shown in SEQ ID NO:1, therebyinactivating the gamma glutamyl hydrolase protein.

Further provided is a molecule capable of binding to one or more ofamino acid residues 110, 171, 220 or 222 in the amino acid sequence ofgamma glutamyl hydrolase as shown in SEQ ID NO:1, wherein the moleculeinactivates gamma glutamyl hydrolase and wherein the molecule has athree dimensional structure complementary to the three dimensionalstructure of gamma glutamyl hydrolase in a fragment that includes one ormore of the one or more of amino acid residues 110, 171, 220 or 222.Compositions comprising the molecule and a suitable carrier, and themolecule and an antifolate, are also provided. The molecule can be usedwith an antifolate to increase the effectiveness of antifolatetreatment.

The molecule can also be used to inactivate gamma glutamyl hydrolaseprotein. Such a method comprises: providing a gamma glutamyl hydrolaseprotein; and exposing the gamma glutamyl hydrolase protein to theabove-described molecule, wherein the molecule binds to one or more ofamino acid residues 110, 171, 220 or 222 in the amino acid sequence ofthe gamma glutamyl hydrolase protein thereby inactivating the gammaglutamyl hydrolase protein.

Further provided is a method of identifying a molecule that inactivatesgamma glutamyl hydrolase protein. The method comprises: determiningwhether a molecule binds to one or more of amino acid residues 110,171,220 or 222 in the amino acid sequence of gamma glutamyl hydrolase asshown in SEQ ID NO:1; and screening a molecule that binds to one or moreof amino acid residues 110, 171, 220 or 222 to determine whether thescreened molecule inactivates gamma glutamyl hydrolase protein. Amolecule identified by the method, as well as a method of inactivatinggamma glutamyl hydrolase using the identified molecule, are alsoprovided.

Further provided is a nucleic acid molecule encoding an inactive gammaglutamyl hydrolase protein, the nucleic acid molecule encoding an aminoacid sequence that substantially corresponds to the amino acid sequenceof native gamma glutamyl hydrolase as shown in SEQ ID NO:1, SEQ ID NO:1being modified at one or more of amino acid residues 110, 171, 220 or222 to render the resulting gamma glutamyl hydrolase protein inactive.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages of this invention will beevident from the following detailed description of preferred embodimentswhen read in conjunction with the accompanying drawings in which:

FIGS. 1A and 1B illustrate SDS 12.5% polyacrylamide gel electrophoresisof purified wildtype and mutant hGH proteins (0.58 μg per well). FIG. 1Ashows a silver stained gel, and FIG. 1B shows a Western blot using apolyclonal antibody (1 to 10,000 dilution) against hGH expressed ininsect cells. 1=wildtype, 2=C19A, 3=C1110A, 4=C124A, 5=C290A;

FIG. 2 illustrates intrinsic fluorescence spectra of wildtype and mutanthGH proteins (0.58 μg/ml in 50 mM sodium acetate pH 5.5, 50 mMβ-mercaptoethanol, 1 mM octyl-β-glucoside). Excitation was at 280 nm.The spectra are numbered as follows: 1=C110A, 2=wildtype, 3=C290A,4=C19A, 5=C124A;

FIG. 3 illustrates the 3-dimensional conformation of gamma glutamylhydrolase, showing the conformational relationship of amino acidresidues 110, 171, 220 and 222; and

FIG. 4 illustrates the conformations of an antigen that is complementaryto the three dimensional structure of an antibody.

DETAILED DESCRIPTION OF THE INVENTION

The cDNA's encoding GH from rat and human sources have been isolated(Yao et al. 1996a; Yao et al. 1996b; U.S. Pat. No. 5,801,031, issuedSep. 1, 1998 and incorporated herein by reference). The hGH cDNA hasbeen expressed in both an insect expression system (Rhee et al. 1998)and Escherichia coli (Yao et al. 1996b). The first 24 amino acidsencoded by the hGH cDNA are a signal peptide, which is removed duringprocessing (Rhee et al. 1998). Therefore, the N-terminal amino acid ofthe mature hGH enzyme is equivalent to R25 in the published hGH sequence(Yao et al. 1996b). As used herein, native gamma glutamyl hydrolaserefers to mature human gamma glutamyl hydrolase protein as described inU.S. Pat. No. 5,801,031, issued Sep. 1, 1998. The mature protein couldbe in vivo or in vitro, and could be isolated from natural sources orsynthesized using protein synthesis technology, including recombinanttechnology. The amino acid sequence of native gamma glutamyl hydrolaseis shown in SEQ ID NO:1. The cDNA encoding native gamma glutamylhydrolase is shown in SEQ ID NO:2. As discussed above, each of SEQ IDNO:1 and SEQ ID NO:2 have the signal peptide removed.

The subject invention provides an inactive gamma glutamyl hydrolaseprotein, the inactive protein having an amino acid sequence thatsubstantially corresponds to the amino acid sequence of native gammaglutamyl hydrolase as shown in SEQ ID NO:1, SEQ ID NO:1 being modifiedat one or more of amino acid residues 110, 171, 220 or 222 to render theresulting gamma glutamyl hydrolase protein inactive. Further provided isa nucleic acid molecule encoding the inactive gamma glutamyl hydrolaseprotein.

As used herein, modified refers to a gamma glutamyl hydrolase proteinhaving reduced catalytic activity in the removal of glutamate fromfolyl- and antifolylpoly-γ-glutamates when compared to the samecatalytic activity of a gamma glutamyl hydrolase protein having an aminoacid sequence as shown in SEQ ID NO:1. Modified refers to elimination ofall catalytic activity (inactive), as well as less than 100% eliminationof catalytic activity (such as a 50% or more reduction in catalyticactivity).

The term “nucleic acid”, as used herein, refers to either DNA or RNA.“Nucleic acid sequence” or “polynucleotide sequence” refers to a single-or double-stranded polymer of deoxyribonucleotide or ribonucleotidebases read from the 5′ to the 3′ end. It includes both self-replicatingplasmids, infectious polymers of DNA or RNA, and nonfunctional DNA orRNA.

“Isolated” nucleic acid refers to nucleic acid which has been separatedfrom an organism in a substantially purified form (i.e. substantiallyfree of other substances originating from that organism), and tosynthetic or recombinantly produced nucleic acid.

The phrase “nucleic acid molecule encoding” refers to a nucleic acidmolecule which directs the expression of a specific protein. The nucleicacid sequences include both the DNA strand sequence that is transcribedinto RNA and the RNA sequence that is translated into protein. The“nucleic acid molecule encoding” includes both the full length nucleicacid sequences as well as non-full length sequences derived from thefull length protein. It being further understood that the “nucleic acidmolecule encoding” further includes degenerate codons of the sequence aswell as sequences which may be introduced to provide codon preference ina specific host cell.

The term “located upstream” as used herein refers to linkage of apromoter upstream from a nucleic acid sequence such that the promotermediates transcription of the nucleic acid sequence.

The term “vector”, refers to viral expression systems, autonomousself-replicating circular DNA (plasmids), and includes both expressionand nonexpression plasmids. Where a recombinant microorganism or cell isdescribed as hosting an “expression vector,” this includes bothextrachromosomal circular DNA and DNA that has been incorporated intothe host chromosome(s). Where a vector is being maintained by a hostcell, the vector may either be stably replicated by the cells duringmitosis as an autonomous structure, or the vector may be incorporatedwithin the host's genome.

The term “plasmid” refers to an autonomous circular DNA molecule capableof replication in a cell, and includes both the expression andnonexpression types. Where a recombinant microorganism or cell isdescribed as hosting an “expression plasmid”, this includes latent viralDNA integrated into the host chromosome(s). Where a plasmid is beingmaintained by a host cell, the plasmid is either being stably replicatedby the cell during mitosis as an autonomous structure, or the plasmid isincorporated within the host's genome.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acid molecules or polynucleotides, orbetween two or more amino acid sequences of peptides or proteins:“reference sequence”, “comparison window”, “sequence identity”,“sequence homology”, “percentage of sequence identity”, “percentage ofsequence homology”, “substantial identity”, and “substantial homology”.A “reference sequence” is a defined sequence used as a basis for asequence comparison; a reference sequence may be a subset of a largersequence, for example, as a segment of a full-length cDNA or genesequence given in a sequence listing or may comprise a complete cDNA orgene sequence.

Optimal alignment of sequences for aligning a comparison window may beconducted, for example, by the local homology algorithm of Smith andWaterman (1981), by the homology alignment algorithm of Needleman andWunsch (1970), by the search for similarity method of Pearson and Lipman(1988), or by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.).

As applied to nucleic acid molecules or polynucleotides or nucleic acidsequences, the term “substantially corresponds to” includes “substantialidentity”, “substantial sequence identity”, “substantial homology”, and“substantial sequence homology”.

As applied to nucleic acid molecules or polynucleotides, the terms“substantial identity” or “substantial sequence identity” mean that twonucleic acid sequences, when optimally aligned (see above), share atleast 90 percent sequence identity, preferably at least 95 percentsequence identity, more preferably at least 96, 97, 98 or 99 percentsequence identity.

“Percentage nucleotide (or nucleic acid) identity” or “percentagenucleotide (or nucleic acid) sequence identity” refers to a comparisonof the nucleotides of two nucleic acid molecules which, when optimallyaligned, have approximately the designated percentage of the samenucleotides. For example, “95% nucleotide identity” refers to acomparison of the nucleotides of two nucleic acid molecules which whenoptimally aligned have 95% nucleotide identity. Preferably, nucleotidepositions which are not identical differ by redundant nucleotidesubstitutions (the nucleotide substitution does not change the aminoacid encoded by the particular codon).

As further applied to nucleic acid molecules or polynucleotides, theterms “substantial homology” or “substantial sequence homology” meanthat two nucleic acid sequences, when optimally aligned (see above),share at least 90 percent sequence homology, preferably at least 95percent sequence homology, more preferably at least 96, 97, 98 or 99percent sequence homology.

“Percentage nucleotide (or nucleic acid) homology” or “percentagenucleotide (or nucleic acid) sequence homology” refers to a comparisonof the nucleotides of two nucleic acid molecules which, when optimallyaligned, have approximately the designated percentage of the samenucleotides or nucleotides which are not identical but differ byredundant nucleotide substitutions (the nucleotide substitution does notchange the amino acid encoded by the particular codon). For example,“95% nucleotide homology” refers to a comparison of the nucleotides oftwo nucleic acid molecules which when optimally aligned have 95%nucleotide homology.

As applied to proteins or polypeptides or amino acid sequences, the term“substantially corresponds to” includes “substantial identity”,“substantial sequence identity”, “substantial homology”, and“substantial sequence homology”.

As applied to proteins or polypeptides, the terms “substantial identity”or “substantial sequence identity” mean that two protein or peptidesequences, when optimally aligned, such as by the programs GAP orBESTFIT using default gap, share at least 90 percent sequence identity,preferably at least 95 percent sequence identity, more preferably atleast 96, 97, 98 or 99 percent sequence identity.

“Percentage amino acid identity” or “percentage amino acid sequenceidentity” refers to a comparison of the amino acids of two proteins orpolypeptides which, when optimally aligned, have approximately thedesignated percentage of the same amino acids. For example, “95% aminoacid identity” refers to a comparison of the amino acids of two proteinsor polypeptides which when optimally aligned have 95% amino acididentity. Preferably, residue positions which are not identical differby conservative amino acid substitutions. For example, the substitutionof amino acids having similar chemical properties such as charge orpolarity are not likely to affect the properties of a protein. Examplesinclude glutamine for asparagine or glutamic acid for aspartic acid.

As further applied to proteins or polypeptides, the terms “substantialhomology” or “substantial sequence homology” mean that two proteins orpeptide sequences, when optimally aligned, such as by the programs GAPor BESTFIT using default gap, share at least 90 percent sequencehomology, preferably at least 95 percent sequence homology, morepreferably at least 96, 97, 98 or 99 percent sequence homology.

“Percentage amino acid homology” or “percentage amino acid sequencehomology” refers to a comparison of the amino acids of two proteins orpolypeptides which, when optimally aligned, have approximately thedesignated percentage of the same amino acids or conservativelysubstituted amino acids. For example, “95% amino acid homology” refersto a comparison of the amino acids of two proteins or polypeptides whichwhen optimally aligned have 95% amino acid homology. As used herein,homology refers to identical amino acids or residue positions which arenot identical but differ only by conservative amino acid substitutions.For example, the substitution of amino acids having similar chemicalproperties such as charge or polarity are not likely to affect theproperties of a protein. Examples include glutamine for asparagine orglutamic acid for aspartic acid.

The phrase “substantially purified” or “isolated” when referring to aprotein (or peptide), means a chemical composition which is essentiallyfree of other cellular components. The protein or peptide can beseparated from an organism or produced synthetically or recombinantly.It is preferably in a homogeneous state although it can be in either adry or aqueous solution. Purity and homogeneity are typically determinedusing analytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A protein (orpeptide) which is the predominant species present in a preparation issubstantially purified. Generally, a substantially purified or isolatedprotein (or peptide) will comprise more than 80% of all macromolecularspecies present in the preparation. Preferably, the protein (or peptide)is purified to represent greater than 90% of all macromolecular speciespresent. More preferably the protein (or peptide) is purified to greaterthan 95%, and most preferably the protein (or peptide) is purified toessential homogeneity, wherein other macromolecular species are notdetected by conventional techniques.

It will be readily understood by those skilled in the art and it isintended here, that when reference is made to particular sequencelistings, such reference includes sequences which substantiallycorrespond to its complementary sequence and those described includingallowances for minor sequencing errors, single base changes, deletions,substitutions and the like, such that any such sequence variationcorresponds to the nucleic acid sequence of the peptide or other proteinto which the relevant sequence listing relates.

The DNA molecules and proteins of the subject invention also includethose analogs, fragments or derivatives which differ from the inactiveGH form in terms of the identity or location of one or more amino acidresidues (deletion analogs containing less than all of the residuesspecified for the protein, substitution analogs wherein one or moreresidues specified are replaced by other residues, and addition analogswherein one or more amino acid residues is added to a terminal or medialportion of the protein) and which share the catalytic activity (thereduction thereof) of the inactive GH. Such analogs, fragments orderivatives will include the modification at one or more of amino acidresidues 110, 171, 220 or 222. At least one of these residues must bemodified from the native form in the inactive form analog, fragment, orderivative. These molecules include, for example: the incorporation ofcodons “preferred” for expression by selected non-mammalian hosts (suchas an N-terminal methionine residue); the provision of sites forcleavage by restriction endonuclease enzymes; and the provision ofadditional initial, terminal or intermediate DNA sequences thatfacilitate construction of readily expressed vectors.

As used herein, a “fragment” of the inactive gamma glutamyl hydrolaseprotein refers to an amino acid sequence of about 10 to about 150 aminoacids. Preferably, the fragments are less than 50 amino acids in length,and more preferably the fragments are 10-20 amino acids in length or20-40 amino acids in length. A fragment as used herein is specificallyintended to include one or more of the modified amino acids residues(residues 110, 171, 220 or 222). Such a fragment is specificallyprovided by the subject invention.

The proteins and fragments thereof described herein can contain anynaturally-occurring or non-naturally-occurring amino acids, includingthe D-form of the amino acids, amino acid derivatives and amino acidmimics, so long as the desired function and activity of theprotein/fragment is maintained. The choice of including an (L)- or a(D)-amino acid in the protein/fragment of the present invention depends,in part, on the desired characteristics of the protein/fragment. Forexample, the incorporation of one or more (D)-amino acids can conferincreased stability on a protein/fragment and can allow aprotein/fragment to remain active in the body for an extended period oftime. The incorporation of one or more (D)-amino acids can also increaseor decrease the pharmacological activity of a protein/fragment.

The protein/fragment may also be cyclized, since cyclization may providethe protein/fragment of the present invention with superior propertiesover their linear counterparts.

As used herein, the terms “amino acid mimic” and “mimetic” mean an aminoacid analog or non-amino acid moiety that has the same or similarfunctional characteristic of a given amino acid. For instance, an aminoacid mimic of a hydrophobic amino acid is one which is non-polar andretains hydrophobicity, generally by way of containing an aliphaticchemical group. By way of further example, an arginine mimic can be ananalog of arginine which contains a side chain having a positive chargeat physiological pH, as is characteristic of the guanidinium side chainreactive group of arginine.

In addition, modifications to the peptide backbone and peptide bondsthereof are also encompassed within the scope of amino acid mimic ormimetic. Such modifications can be made to the amino acid, derivativethereof, non-amino acid moiety or the peptide either before or after theamino acid, derivative thereof or non-amino acid moiety is incorporatedinto the protein. What is critical is that such modifications mimic thepeptide backbone and bonds which make up the same and have substantiallythe same spacial arrangement and distance as is typical for traditionalpeptide bonds and backbones. An example of one such modification is thereduction of the carbonyl(s) of the amide peptide backbone to an amine.A number of reagents are available and well known for the reduction ofamides to amines such as those disclosed in Wann et al. (1981) andRaucher et al. (1980). An amino acid mimic is, therefore, an organicmolecule that retains the similar amino acid pharmacophore groups as arepresent in the corresponding amino acid and which exhibits substantiallythe same spatial arrangement between functional groups.

The substitution of amino acids by non-naturally occurring amino acidsand amino acid mimics as described above can enhance the overallactivity or properties of an individual protein/fragment thereof basedon the modifications to the backbone or side chain functionalities. Forexample, these types of alterations can enhance the protein's/fragment'sstability to enzymatic breakdown and increase biological activity.Modifications to the peptide backbone similarly can add stability andenhance activity.

One skilled in the art, using the above sequences or formulae, caneasily synthesize the proteins/fragments of this invention. Standardprocedures for preparing synthetic peptides are well known in the art.The novel peptides can be synthesized using: the solid phase peptidesynthesis (SPPS) method of Merrifield (1964) or modifications of SPPS;or, the peptides can be synthesized using standard solution methods wellknown in the art (see, for example, Bodanzsky (1993)). Alternatively,simultaneous multiple peptide synthesis (SMPS) techniques well known inthe art can be used. Peptides prepared by the method of Merrifield canbe synthesized using an automated peptide synthesizer such as theApplied Biosystems 431A-01 Peptide Synthesizer (Mountain View, Calif.)or using the manual peptide synthesis technique described by Houghten(1985).

The inactive GH of the subject invention is inactivated by modifying oneor more of amino acid residues 110, 171, 220 or 222 in SEQ ID NO:1. Theone or more amino acid residues can be modified by any means known inthe art. Site directed mutagenesis of a nucleic acid molecule whichencodes the amino acid sequence can be used to substitute another aminoacid for the native amino acid. Antibodies, small molecules, or shortpeptides could be used which bind to and block the residue (therebyinterfering with the 3-dimensional conformation of the active protein).Both of these scenarios are intended to be included as a “modified”amino acid.

The invention thus further provides a method of inactivating gammaglutamyl hydrolase protein. The method comprises providing a gammaglutamyl hydrolase protein, and modifying one or more of amino acidresidues 110, 171, 220 or 222 in the amino acid sequence of the gammaglutamyl hydrolase protein as shown in SEQ ID NO:1 (thereby inactivatingthe gamma glutamyl hydrolase protein).

A molecule capable of binding to one or more of amino acid residues 110,171, 220 or 222 in the amino acid sequence of gamma glutamyl hydrolaseas shown in SEQ ID NO:1 is also provided, wherein the moleculeinactivates gamma glutamyl hydrolase and wherein the molecule has athree dimensional structure complementary to the three dimensionalstructure of gamma glutamyl hydrolase in a fragment that includes one ormore of the one or more of amino acid residues 110, 171, 220 or 222. Thethree dimensional structure of gamma glutamyl hydrolase is shown in FIG.3, including the location of residues 110, 171, 220, and 222. Theconcept of a complementary three dimensional structure is clearlyillustrated in FIG. 4, which shows the conformations of an antigen (10)complementary to the three dimensional structure of an antibody (12).

As should be readily apparent to those skilled in the art, the isolatedmolecule could be, for example, an antibody (such as a polyclonal ormonoclonal antibody, including chimeric or humanized antibodies), apeptide (of about 10 to about 100 amino acids in length, preferably lessthan 50 and most preferably 10-20 or 20-10 40 amino acids in length), orother small molecule capable of binding to one or more of amino acidresidues 110, 171, 220 or 222. By “binding to” is meant covalent ornon-covalent attachment to the particular amino acid residue orotherwise blocking of the particular amino acid residue. For example,amino acid residue 110 could be bound by an antibody that recognizes anepitope that includes amino acid residue 110. A small molecule whichcovalently attaches to amino acid residue 110 also “binds” that residue110. Alternatively, a small molecule which covalently attaches to aminoacid residues 109 and 111, effectively sterically blocking amino acidresidue 110, is considered to “bind” amino acid residue 110 as usedherein. Bind is therefore used in the broader sense of blockage of threedimensional conformation of the particular amino acid.

Suitable molecules capable of binding to one or more of amino acidresidues 110, 171, 220 or 222 can be identified by any means known inthe art. For example, a peptide can be synthesized which includes aminoacid residues 105-115 of SEQ ID NO:1. The chemically synthesized peptidecan be conjugated to bovine serum albumin and used for raisingpolyclonal antibodies in rabbits. Standard procedures can be used toimmunize the rabbits and to collect serum, as described below.Polyclonal antibody can be tested for its ability to bind to GH (or theGH fragment 105-115). For polyclonal antibody that shows a high affinitybinding to GH, functional studies can then be undertaken for reductionin GH catalytic activity. Fragments (such as Fab, Fc, F(ab′)₂) of thepolyclonal antibody can be made if steric hindrance appears to bepreventing an accurate evaluation of more specific modulating effects ofthe antibody (Becker and Miller 1989, Kupinski and Miller 1986, andMiller et al. 1986). Polyclonal antibody to the synthetic peptide thatrecognizes GH and reduces GH catalytic activity can be obtained at ≧95%purity and conjugated to bovine serum albumin or to another carrierprotein, for the production of murine monoclonal antibodies.

Alternatively, monoclonal antibody production can be carried out usingBALB/c mice. Immunization of B-cell donor mice can involve immunizingthem with antigens mixed in TiterMax™ adjuvant as follows: 50 μgantigen/20 μl emulsion×2 injections given by an intramuscular injectionin each hind flank on day 1. Blood samples can be drawn by tail bleedson days 28 and 56 to check the titers by ELISA assay. At peak titer(usually day 56) the mice can be subjected to euthanasia by CO₂inhalation, after which splenectomies can be performed and spleen cellsharvested for the preparation of hybridomas by standard methods.

Once a monoclonal antibody has been identified which binds to one ormore of amino acid residues 110, 171, 220 or 222, bacteriophage displaylibraries can be used to identify peptide molecules which mimic themonoclonal antibody conformation. Such identified peptides can then beused in turn to identify peptide molecules that bind to the originalamino acid residues 110, 171, 220 or 222 (the concept of mimeticcompounds).

Scott and Smith (1990) presented a method of defining peptide ligands byusing randomly synthesized peptide inserts in bacteriophage. Relatedmethods were published by Cwirla et al. (1990) and by Devlin et al.(1990). Since that time a literature has arisen in which both theoriginal hexapeptide inserts and larger inserts have been used inidentifying epitopes recognized by monoclonal antibodies. For example, awell-balanced decapeptide (10-mer) library (described by Christian etal. 1992) or a dodecapeptide (12-mer) library (Clontech Laboratories,Palo Alto, Calif.) can be used. The strategy for using these librarieslargely follows the review presented by Scott (1992) and employs, withmodifications, the detailed methodology for use of this system asdescribed by Smith and Scott (1993). A useful strategy is describedbelow in the Materials and Methods.

Having thus identified molecules capable of binding to one or more ofamino acid residues 110, 171, 220 or 222, tissues or cells could becontacted with compositions of the molecules in order to inactivategamma glutamyl hydrolase and/or to increase the effectiveness ofantifolate treatment. In the context of this invention, to “contact”tissues or cells with a composition means to add the composition,usually in a suitable liquid carrier, to a cell suspension or tissuesample, either in vitro or ex vivo, or to administer the composition tocells or tissues within an animal (including humans). In one embodiment,the composition may comprise the molecule and the anti-folate togetheras one composition (mixed or attached to one another by any means knownin the art, including covalent or non-covalent attachment or otherbinding). By contacting the tissues or cells with the compositions ofthe molecules, the gamma glutamyl hydrolase protein present in thetissues or cells is thereby exposed to the molecule.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill in the art. Ingeneral, for therapeutics, a patient suspected of needing such therapyis given a composition in accordance with the invention, commonly in apharmaceutically acceptable carrier, in amounts and for periods whichwill vary depending upon the nature of the particular disease, itsseverity and the patient's overall condition. The pharmaceuticalcompositions of the present invention may be administered in a number ofways depending upon whether local or systemic treatment is desired andupon the area to be treated. Administration may be topical (includingophthalmic, vaginal, rectal, intranasal, transdermal), oral orparenteral. Parenteral administration includes intravenous drip orinfusion, subcutaneous, intraperitoneal or intramuscular injection,pulmonary administration, e.g., by inhalation or insufflation, orintrathecal or intraventricular administration.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

Compositions for parenteral, intrathecal or intraventricularadministration may include sterile aqueous solutions which may alsocontain buffers, diluents and other suitable additives.

In addition to such pharmaceutical carriers, cationic lipids may beincluded in the formulation to facilitate uptake. One such compositionshown to facilitate uptake is Lipofectin (BRL, Bethesda Md.).

Dosing is dependent on severity and responsiveness of the condition tobe treated, with course of treatment lasting from several days toseveral months or until a cure is effected or a diminution of diseasestate is achieved. Optimal dosing schedules can be calculated frommeasurements of drug accumulation in the body. Persons of ordinary skillcan easily determine optimum dosages, dosing methodologies andrepetition rates. Optimum dosages may vary depending on the relativepotency of individual compositions, and can generally be calculatedbased on IC₅₀'s or EC₅₀'s in in vitro and in vivo animal studies. Forexample, given the molecular weight of compound and an effective dosesuch as an IC₅₀, for example (derived experimentally), a dose in mg/kgis routinely calculated.

The subject invention thus also provides a method of inactivating gammaglutamyl hydrolase protein. The method comprises providing a gammaglutamyl hydrolase protein, and exposing the gamma glutamyl hydrolaseprotein to the molecule described above. The molecule binds to one ormore of amino acid residues 110, 171, 220 or 222 in the amino acidsequence of the gamma glutamyl hydrolase protein, thereby inactivatingthe gamma glutamyl hydrolase protein.

As indicated above, the molecules according to the subject invention areparticularly useful to increase the effectiveness of antifolatetreatment. Such a method is provided by the subject invention, andcomprises co-administering the molecule and an antifolate.Co-administering is intended to cover simultaneous (as one combined oras two simultaneously administered separate compositions) or sequentialadministration of the molecule and the antifolate separately.

As used herein, “antifolatel” refers to antifolates which are convertedto polyglutamates (specifically, antifolylpolyglutamates) in host ortumor tissues. Examples of antifolates as used herein include, forexample, methotrexate, aminopterin, and more recently developedantifolates such as edetrexate, lomotrexol, BW1843U89, and ZD1694(Fleming and Schilsky 1992; Bertino 1993). The resultingantifolylpolyglutamates are then degraded by gamma glutamyl hydrolaseback to the parent compound (the antifolate) and glutamic acid. Ingeneral, the antifolylpolyglutamates are more toxic due to their greatercellular retention and tighter binding to a drug target than theantifolates.

The invention also provides a method of identifying a molecule thatinactivates gamma glutamyl hydrolase protein. The method comprisesdetermining whether a molecule binds to one or more of amino acidresidues 110,171, 220 or 222 in the amino acid sequence of gammaglutamyl hydrolase as shown in SEQ ID NO:1, and screening a moleculethat binds to one or more of amino acid residues 110, 171, 220 or 222 todetermine whether the screened molecule inactivates gamma glutamylhydrolase protein. Examples of such a method are described in furtherdetail above in the context of identification of antibody or peptidemolecules which bind to one or more of amino acid residues 110, 171, 220or 222.

If the molecule is a peptide, phage display libraries can be used todetermine whether the molecule binds to the peptide fragment whichincludes the amino acid residue. If the molecule is an antibody, theantibody can be immobilized on a solid support and the peptide fragmentwhich includes the amino acid residue can be labeled with a detectablemarker and contacted with the immobilized antibody. After washing, thepresence of the label will indicate that the antibody bound to thepeptide. Likewise, the peptide could be immobilized and the antibodycould be contacted with the immobilized peptide. These techniques arereadily known in the art.

Once a suitable molecule has been identified, which molecule is alsoprovided by the subject invention, the molecule can be used toinactivate gamma glutamyl hydrolase protein or to increase theeffectiveness of antifolate treatment.

The subject invention also provides a nucleic acid molecule encoding aninactive gamma glutamyl hydrolase protein, the nucleic acid moleculeencoding an amino acid sequence that substantially corresponds to theamino acid sequence of native gamma glutamyl hydrolase as shown in SEQID NO:1, SEQ ID NO:1 being modified at one or more of amino acidresidues 110, 171, 220 or 222 to render the resulting gamma glutamylhydrolase protein inactive. Preferably, the nucleic acid molecule has anucleic acid sequence that substantially corresponds to the nucleic acidsequence of native gamma glutamyl hydrolase protein as shown in SEQ IDNO:2, SEQ ID NO:2 being modified at one or more of nucleotides 328-330,511-513, 658-660, or 664-666 (the nucleotides which encode amino acidresidues 110, 171, 220, and 222, respectively).

The nucleic acid molecule can be deoxyribonucleic acid (DNA) orribonucleic acid (RNA, including messenger RNA or mRNA), genomic orrecombinant, biologically isolated or synthetic. The DNA molecule can bea cDNA molecule, which is a DNA copy of a messenger RNA (mRNA) encodingthe inactivated GH.

The nucleic acid molecules of the subject invention can be expressed insuitable host cells using conventional techniques. Any suitable hostand/or vector system can be used to express the inactive GH. Bacterialhosts (for example, Escherichia coli) and mammalian hosts (for example,Hela cells, Cv-1 cells, COS cells) are preferred. In tumor host cells,where the hydrolysis effect of GH on antifolylpolyglutamates decreasesthe efficiency of the antifolate treatment, it is desirable to decreaseor prevent expression of native GH. Thus, tumor cells are a particularlysuitable host in which to decrease native GH expression such as byexpression of the inactive GH of the subject invention instead of nativeGH.

Techniques for introducing the nucleic acid molecules into host cellsmay involve the use of expression vectors which comprise the nucleicacid molecules. These expression vectors (such as plasmids and viruses;viruses including bacteriophage) can then be used to introduce thenucleic acid molecules into suitable host cells. For example, DNAencoding the inactive GH can be injected into the nucleus of a host cellor transformed into the host cell using a suitable vector, or mRNAencoding the inactive GH can be injected directly into the host cell, inorder to obtain expression of inactive GH in the host cell.

Various methods are known in the art for introducing nucleic acidmolecules into host cells. One method is microinjection, in which DNA isinjected directly into the nucleus of cells through fine glass needles(or RNA is injected directly into the cytoplasm of cells).Alternatively, DNA can be incubated with an inert carbohydrate polymer(dextran) to which a positively charged chemical group (DEAE, fordiethylaminoethyl) has been coupled. The DNA sticks to the DEAE-dextranvia its negatively charged phosphate groups. These large DNA-containingparticles stick in turn to the surfaces of cells, which are thought totake them in by a process known as endocytosis. Some of the DNA evadesdestruction in the cytoplasm of the cell and escapes to the nucleus,where it can be transcribed into RNA like any other gene in the cell. Inanother method, cells efficiently take in DNA in the form of aprecipitate with calcium phosphate. In electroporation, cells are placedin a solution containing DNA and subjected to a brief electrical pulsethat causes holes to open transiently in their membranes. DNA entersthrough the holes directly into the cytoplasm, bypassing the endocytoticvesicles through which they pass in the DEAE-dextran and calciumphosphate procedures. DNA can also be incorporated into artificial lipidvesicles, liposomes, which fuse with the cell membrane, delivering theircontents directly into the cytoplasm. In an even more direct approach,DNA is absorbed to the surface of tungsten microprojectiles and firedinto cells with a device resembling a shotgun.

Several of these methods, microinjection, electroporation, and liposomefusion, have been adapted to introduce proteins into cells. For review,see Mannino and Gould-Fogerite 1988, Shigekawa and Dower 1988, Capecchi1980, and Klein et al. 1987.

Further methods for introducing nucleic acid molecules into cellsinvolve the use of viral vectors. Since viral growth depends on theability to get the viral genome into cells, viruses have devised cleverand efficient methods for doing it. One such virus widely used forprotein production is an insect virus, baculovirus. For a review ofbaculovirus vectors, see Miller (1989). Various viral vectors have alsobeen used to transform mammalian cells, such as bacteriophage, vacciniavirus, adenovirus, and retrovirus.

As indicated, some of these methods of transforming a cell require theuse of an intermediate plasmid vector. U.S. Pat. No. 4,237,224 to Cohenand Boyer describes the production of expression systems in the form ofrecombinant plasmids using restriction enzyme cleavage and ligation withDNA ligase. These recombinant plasmids are then introduced by means oftransformation and replicated in unicellular cultures includingprocaryotic organisms and eucaryotic cells grown in tissue culture. TheDNA sequences are cloned into the plasmid vector using standard cloningprocedures known in the art, as described by Sambrook et al. (1989).

With this understanding of the scope of the subject invention, thedetails which follow describe the identification of the catalytic(active) site of human gamma glutamyl hydrolase (GH), as well we theelucidation of the 3-dimensional structure of GH in the region(fragment) that includes the catalytic site. Specifically, usingsite-directed mutagenesis the cDNA for human GH was altered to encodefour different proteins each with one of four cysteine residues changedto alanine. Three of the mutant proteins had activities similar towildtype GH and were inhibited by iodoacetic acid whereas the C110Amutant had no activity. C110 is conserved among the human, rat and mouseGH amino acid sequences. The wildtype protein and all four mutants hadsimilar intrinsic fluorescence spectra indicating no major structuralchanges had been introduced. These results indicate that C110 iscatalytically essential and suggest that GH is a cysteine peptidase.

Using sensitive sequence analysis methods to extract subtle patternsfrom sequence databases, a statistical significance similarity was foundbetween human gamma glutamyl hydrolase (hGH) and the class-I glutamineamidotransferase family of enzymes. In particular, the catalytic activesite from the latter is conserved in hGH as well as other amino acidsnear the catalytic residues. This leads to the conclusion that hGH foldssimilar to the class-I glutamine amidotransferases. Referring to FIG. 3,the 3-dimensional model predicts that Cys110 functions as the activesite nucleophile attacking the γ-carbonyl of glutamine to form aglutamyl thioester intermediate (Thoden et al. 1998). The model alsopredicts that His 220 and Glu 222 in hGH are the other two amino acidsin the catalytic triad. His 220 and Glu 222 are conserved in the human,rat and mouse glutamyl hydrolase sequences. In the proposed model forhGH, His 220 would activate Cys 110 and Glu 222 would stabilize theresulting positively charged imidizolium cation. The alignment modelalso predicts that His 171 points away from the catalytic triad and isinvolved in substrate binding not catalysis.

Materials and Methods

Construction of C19A, C110A, C124A, and C290A Mutants of hGH

A cDNA encoding the 294 amino acid mature form of hGH protein with anN-terminal methionine preceding the first arginine residue hadpreviously been subcloned into pET-24a (pET-hGH, Yao et al. 1996b)(Novagen, Madison, Wis.). Site-directed mutagenesis was performed withthe QuickChange kit (Stratagene, La Jolla, Calif.) according to themanufacturer's protocol using 125 ng of each primer for the C110Amutation and 150 ng for the other mutations, and annealing temperaturesof 55° C. for C110A and 53° C. for the other mutations. Theoligonucleotides synthesized for the mutagenesis were as follows, (codonchanged is underlined and mismatched basepairs are in bold).

GHC19+ (SEQ ID NO:3) 5′-GCC CAT CAT CGG AAT ATT AAT GCA AAA AGC CCG TAATAA AGT C-3′

GHC19− (SEQ ID NO:4) 5′-GAC TTT ATT ACG GGC TTT TTG CAT TAA TAT TCC GATGAT GGG C-3′

GHC110+ (SEQ ID NO:5) 5′-CCT GTG TGG GGC ACA GCG CTT GGA TTT GAA GAGC-3′

GHC110− (SEQ ID NO:6) 5′-GCT CTT CAA ATC CAA GCG CTG TGC CCC ACA CAGG-3′

GHC124+ (SEQ ID NO:7) 5′-GCT GAT TAG TGG AGA GGC CTT ATT AAC TGC CACAG-3′

GHC124− (SEQ ID NO:8) 5′-CTG TGG CAG TTA ATA AGG CCT CTC CAC TAA TCAGC-3′

GHC290+ (SEQ ID NO:9) 5′-CTT CAT TTC AGC AAG CTT ACA TAT TTG ATT GAA AGTC-3′

GHC290− (SEQ ID NO:10) 5′-GAC TTT CAA TCA AAT ATG TAA GCT TGC TGA AATGAA G-3′

Plasmid DNA was purified using the Plasmid Midi kit (Qiagen, SantaClarita, Calif.). The sequences of all mutant clones were confirmedusing automated DNA sequencing on either-an Applied Biosystems 373A or377A DNA sequencer (Perkin Elmer, Branchburg, N.J.).

Expression and Purification of Wildtype and Mutant hGH

Mutant plasmids were used to transform E. colistrain BL21(DE3)pLysS tokanamycin resistance. Cultures (20 ml) were grown for 16 hours at 37°C., 225 rpm in tryptone-phosphate broth (Moore et al. 1993), containing30 μg/ml kanamycin and 34 μg/ml chloramphenicol. An aliquot (6 ml) ofthis culture was used to inoculate 500 ml of the same medium. Cultureswere incubated at 27° C., 225 rpm until the OD_(600nm) was 0.5-0.6.Isopropyl β-D-thiogalactoside (IPTG) was added to a final concentrationof 1 mM and cultures were incubated for a further 3 hours. Cells wereharvested by centrifugation and stored at −80° C. Cells from 1 liter ofbroth were resuspended in 50 ml of 50 mM Tris/HCl pH 7.5 buffercontaining 500 mM NaCl and Complete protease inhibitor cocktail(Boehringer Mannheim, Indianapolis, Ind.). The mixture was sonicated andclarified by centrifugation at 20,000 g for 30 minutes at 4° C. A 20-60%ammonium sulfate precipitate was resuspended in 50 mM sodium acetate, pH5.8, 1 mM octyl-β-D-glucoside (OBG) and 5% glycerol (buffer A). Afterdialysis at 4° C. against buffer A and clarification by centrifugationat 30,000 g, 4° C. for 120 minutes, the dialysate was applied in twoaliquots to a Protein Pak SP colummn (1.0 cm×10 cm, Waters Division ofMillipore Corp., Milford, Mass.) equilibrated in buffer A. The proteinwas eluted (1 ml/min) with a linear gradient in buffer A of 0 to 1M NaClover 60 minutes. Fractions were assayed for hGH activity or for hGHprotein by Western blot. Fractions containing hGH were pooled and frozenat −80° C. Wildtype hGH and the C19A, C110A, and C290A mutants eluted astwo peaks whereas the C124A mutant eluted as a single peak coincidentwith the first peak for the other hGH proteins. Western blottinganalysis indicated that the hGH in both peaks was of the same Mr. Therewas 8-10 times more hGH protein in peak 1 than in peak 2. Astatistically significant amount (40-82%) of enzyme activity of proteinspurified on the SP column was lost when stored either at 4° C. or at−80° C. Thawed aliquots of peak 1 were incubated for 2 hours at 4° C.with 2 ml of pHMB agarose (0.8 μmole/ml agarose) (Sigma Chemical, St.Louis, Mo.) equilibrated in 50 mM sodium acetate pH 5.8, 20 mM OBG, 500mM NaCl (buffer B). The matrix was poured into a column (0.75 cm×4.5 cm)and washed with buffer B. hGH protein was eluted with buffer Bcontaining additionally 10 mM dithiothreitol and 50 mMβ-mercaptoethanol. EDTA (1 mM final) was added to each fraction toinhibit remaining proteases. Fractions containing hGH were pooled andstored at −80° C.

Protein Concentration Assay

Protein concentrations were determined using the Coomassie Plus proteinassay reagent (Pierce, Rockford, ill.) with bovine serum albumin as thestandard. The concentrations of purified wildtype and mutant hGHproteins were normalized according to the intensity of their intrinsicfluorescence spectra relative to the spectra of C110A (the most pureprotein).

Activity Assays

hGH activity in chromatography fractions was measured with4-NH₂-10-CH₃PteGlu₅ (Schirk Labs, Jona, Switzerland) as previouslydescribed (Rhee et al. 1998). The specific activities and reactionkinetics of hGH were determined using varying concentrations of4-NH₂-10-CH₃PteGlu₂ as substrate (Schirk Labs). K_(m) and V_(max) weredetermined by the method of Hanes (Price and Stevens 1989) with the bestfit line calculated using Excel (Microsoft). Iodoacetate inhibition wasperformed as previously described using 0.5 mM iodoacetate for 60minutes (Rhee et al. 1998).

SDS-PAGE

Proteins were separated using 12% (4% stacking) SDS-PAGE gels. Proteinbands were visualized by silver staining (Bio-Rad, Hercules, Calif.),following the manufacturer's protocol.

Western Blottinq

hGH protein was determined by Western blotting as previously described(Rhee et al. 1998).

Measurement of Intrinsic Fluorescence

Intrinsic fluorescence of wildtype and mutant hGH proteins was measuredon a Perkin Elmer LS-50B fluorescence spectrometer.

Biopanning of Monoclonal Antibody with Bacteriophage Display Libraries

In the first round of biopanning a 60 mm streptavidin-coated petri dishis filled with blocking solution (0.5% BSA, 0.1 M NaHCO₃₁ 0.1 μg/mlstreptavidin, 0.2% NaN₃) for 2 hours, then washed three times withTBS-0.5% Tween. Next, 1 μl of the library (about 1×10¹¹ phage) that hasbeen incubated overnight at 4° C. with 1 μg of biotinylated Mab isdiluted with 1 ml of TBS-Tween, and this mixture is then added to thepetri dish and rocked for 15 minutes at room temperature. The petri dishis washed 10 times with TBS-Tween, and bound phage is eluted bypipetting 800 μl of 0.1 N HCl (pH adjusted to 2.2 with glycine)—1 mg/mlBSA into the dish. The eluate is then pipetted into a microfuge tubecontaining 48 μl of 2M Tris, to bring the pH up to about 8.

The eluate is concentrated and washed twice in TBS using an AmiconCentricon-30 filter (Amicon, Inc., Beverly, Mass.). This final productis titered out by making dilutions from a small amount of concentratedeluate in TBS-0.1% gelatin and adding 1 μl of each dilution made to 19μl of TBS-gelatin, then adding 20 μl of starved K91 E. coli cells andincubating for 10 minutes at room temperature. After adding 200 μl ofNZY medium containing 0.2 μg/ml tetracycline (Tc) and incubating at 37°C. for 1 hour, the mixture is plated out on NZY agar plates containing40 μg/ml tetracycline and allowed to grow up overnight at 37° C.

After titering, the entire concentrated eluate from the first round ofbiopanning (about 50 μl) is added to an equal volume of fresh starvedK91 cells, and amplification performed as described by Smith and Scott(1993). Following the first PEG/NaCl precipitation, the resulting pelletis dissolved in 1 ml TBS. Phage is then precipitated a second time withPEG/NaCl, allowed to stand at least 1 hour at 4° C., and the precipitatecollected following centrifugation at 4° C. After careful removal of allthe supernatant, the pellet is dissolved in 100 μl TBS. This amplifiedproduct can then be titered.

The second biopanning also uses 1 μg of biotinylated antibody with1×10¹¹ phage, and the second round of biopanning is concentrated andamplified as in the first round. In the third round, 0.01 μg ofbiotinylated antibody is biopanned against 2.5×10¹¹ phage. The thirdround is stopped after eluting the bound phage from the petri dish. Thiseluate is not concentrated or amplified. Titerings are done before andafter each round, and the percent yield is calculated as the number ofbacteriophage obtained in an elution fraction relative to the initialnumber of bacteriophage (Christian et al. 1992). A yield shouldgenerally be greater than 10⁻⁵ to exceed background, with values of 10⁻⁴to 10⁻¹ typically observed. Increasing percent yields in subsequentrounds of biopanning are, in particular, suggestive that clones ofincreasing affinity are being selected.

In some experiments, an immunological screening assay, as described byChristian, et al. (1992) may be performed using NZY+Tc agar platescontaining about 500 well-separated colonies. The colonies aretransferred to nitrocellulose membrane filters (Biorad Laboratories,Hercules, Calif.), and the filters are immediately washed twice in TNTBuffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20), blocked for 30minutes at room temperature with gentle agitation in 20% normal goatserum in TNT buffer, then incubated for 2 hours at room temperature inprimary mab that has been diluted 1:1000 in blocking buffer. The filtersare washed sequentially for 10 minutes at room temperature each wash, inwashing buffer A (TNT Buffer +0.1% BSA), washing buffer B (TNTBuffer+0.1% BSA+0.1% NP-40), and then again washing buffer A, andincubated in a secondary peroxidase-conjugated goat anti-mouse IgG for1½ hours at room temperature. The filters are washed as before, then putin a final wash of TN (10 mM Tris, pH. 7.5, 150 mM NaCl). Colordevelopment is observed after putting filters in ABTS substrate.

Small cultures of individual colonies are then grown up overnight, byeither: a) selecting the colonies that were positive from theimmunological screening; or b) skipping the screening step and randomlyselecting colonies (about 100). Each colony is inoculated into 2 ml ofNZY medium containing 20 μg/ml tetracycline, and these small culturesgrown up overnight at 37° C., with vigorous shaking. The next daycultures are centrifuged to pellet the cells, and the supernatant isremoved. To 1 ml of the supernatant is then added 150 μl PEG/NaCl, andthe phage are precipitated overnight at 4° C. Following subsequentcentrifugation and removal of supernatant, the pellet is dissolved in 1ml TBS.

For DNA sequencing, 400 μl of the dissolved pellet is extracted oncewith phenol, and the resulting aqueous phase (about 300 μl) is added to500 μl TE and 80 μl 3M sodium acetate buffer. Then 1 ml ethanol is addedand the SS DNA is allowed to precipitate overnight at 4° C. Each sampleis then microfuged for 30 minutes at 4° C., the DNA pellet washed oncein 70% ETOH, dried, and resuspended in 7 μl H₂O. This template can bestored at −20° C. until ready to use.

Due to the quite GC-rich Sfi I cloning site flanking the insertionregion (Christian et al. 1992), sequencing reactions are carried outusing the Sequenase 7-deaza dGTP DNA sequencing kit (Amersham-USBiochemicals, Arlington Heights, Ill.) with ³²P-dATP and an antisenseprimer located approximately 40 nucleotides 3′ to the insert site.Samples are run on a standard 6% sequencing gel using an IBI STS 45sequencing apparatus (Eastman Kodak Company, Rochester, N.Y.). The GCGsoftware (Genetics Computer Group, Inc., Madison Wis.) is helpful foraligning sequences obtained from multiple clones in order to findconsensus sequences.

EXAMPLE I Expression and Purification of Wildtype and Mutant hGHProteins

Site-directed mutagenesis was used to generate C19A, C110A, C124A, andC290A variants of hGH, each having 1 cysteine codon replaced by analanine codon. Wildtype and mutant proteins were expressed inEscherichia coli using the pET expression system (Studier and Moffatt1986). Wildtype hGH and the C19A, C110A, C124A, and C290A mutants wereinitially purified on a SP cation exchange column. In the case of theC110A mutant no fractions contained hGH activity.

The SP purified proteins were further purified on a column of pHMBlinked to agarose (Waltham et al. 1997). All four cysteine mutantproteins bound to pHMB coupled agarose indicating that there was morethan one cysteine residue in hGH, which bound this matrix. Using thisprocedure wildtype hGH was isolated with an overall yield of 11%.Analysis by SDS-PAGE and Western blotting of pooled fractions indicatedhGH was purified to near homogeneity (FIG. 1). The Mr of the major bandat 35 kDa is in agreement with the theoretical mass of the cDNA encodedprotein (Yao et al. 1996b). In most preparations there was a minornon-immunoreactive protein that copurified with hGH and had afractionally lower Mr.

EXAMPLE II Activity of Wildtype and Mutant hGH Proteins

The specific activities of the purified hGH proteins are summarized inTable 1. Wildtype, C19A, C124A, and C290A hGH proteins had activity andall produced a similar product distribution of methotrexatepolyglutamates when 4-NH₂-10-CH₃PteGlu, was used as a substrate. Thespecific activities of C19A and C124A were significantly lower than forthe wildtype protein but these proteins had a higher amount ofcontaminating protein (FIG. 1). The activities of wildtype, C19A, C124A,and C290A proteins were reduced 70-94% by incubation with 0.5 mMiodoacetate for 60 minutes. The fact that the C19A, C124A and C290Amutant proteins were inhibited by iodoacetate suggests that all thesemutant proteins still contain a catalytically essential cysteineresidue, namely C110.

The K_(m) and V_(max) for the active hGH proteins with4-NH₂-10-CH₃PteGlu₂ were determined (Table 1). The V_(max) values forthe wildtype and active mutants were not significantly different,indicating that the differences in specific activities of the purifiedproteins (Table 1) were due to an increased amount of contaminantprotein. The Km values for 4-NH₂-10-CH₃PteGlu₂ with the C19A, C124A, andC290A mutant hGH proteins were significantly lower than for wildtypehGH, indicating a higher affinity for this substrate. The C110A mutanthad no activity (less than 0.01% of wildtype enzyme) when assayed witheither 4-NH₂-10-CH₃PteGlu₂ or the better substrate, 4-NH₂-10-CH₃PteGlu.

An amino acid sequence alignment of the human (Yao et al. 1996b), rat(Yao et al. 1996a) and mouse (Esaki et al. 1998) GH proteins indicatedthe C19 and C110 were invariant in all three sequences (Table 2). Sincethe C19A mutant had a V_(max) similar to the wildtype hGH, C19 cannot becatalytically essential. There are no equivalents of C124 or C290 in rator mouse GH proteins. Therefore, it is unlikely that either is involvedin catalysis.

The C19A, C124A and C290A proteins all had increased affinities for4-NH₂-10-CH₃PteGlu₂. Although hGH cleaves both at the carboxyl terminaland penultimate γ-glutamyl bond, the penultimate bond is favored (Rheeet al. 1998). It has also been demonstrated that 4-NH₂-10-CH₃PteGlu₂ isa poor substrate for hGH relative to longer chain polyglutamates (Rheeet al. 1998); Rhee et al. 1995), presumably because hydrolysis can occuronly at the terminal γ-glutamyl bond. Therefore, the increased affinityfor 4-NH₂-10-CH₃PteGlu₂ by the active mutants may be a feature of thisparticular substrate. Given the complex nature of cleavage of substrateswith longer chain lengths, it is currently impossible to obtain detailedkinetic data for these more favored substrates. The similar productdistribution shown when the active mutants were assayed with4-NH₂-10-CH₃PteGlu₅, favoring cleavage at the penultimate γ linkage(Rhee et al. 1998) suggests that these mutations had no effect on thespecificity of the location of the γ-bond cleavage.

The loss of activity when C110 is mutated to alanine and the fact thatC110 is conserved in the amino acid sequences of human, rat and mouse GHindicates that this is the essential cysteine.

EXAMPLE III Intrinsic Fluorescence of hGH and Mutant Proteins

Wildtype and mutant hGH proteins were diluted to the same concentrationof total protein and the intrinsic fluorescence spectra of theseproteins were measured. Excitation at 280 nm produced emission spectrathat were identical in shape and λ_(max) for all the purified hGHproteins (FIG. 2). The intrinsic fluorescence spectra were alsoidentical to that measured for hGH expressed in insect cells.Differences in fluorescence intensity could be attributed to the slightdifferences in purity between the preparations as seen by silver stainedSDS-PAGE and Western blotting (FIG. 1) and specific activities (Table1). Emission spectra had a fluorescence intensity peak at 337-338 nm.Although this method cannot detect very small structural changes itsuggests that there were no major structural changes caused bymutagenesis.

EXAMPLE IV 25 Three-dimensional Model for the Catalytic Site of HumanGamma Glutamyl Hydrolase

Many enzymes involved in producing precursors for DNA synthesis requirefolate as a cofactor. Antifolate drugs, for example, methotrexate, whichimpair folate function, are the primary treatments for many cancers. Theretention and efficacy of folates and antifolate drugs within the cellare dependent on the addition of a poly-γ-glutamate chain to themonoglutamate. Folylpolyglutamate synthetase (FPGS) catalyzes thesequential addition of glutamate and y-glutamyl hydrolase (GH) catalyzesthe removal of glutamate from folyl and antifolyl poly-γ-glutamates. Thebalance between GH and FPGS activity regulates the amount ofglutamylation of folate and antifolate drugs in the cell. The use of lowmolecular weight inhibitors of GH in conjunction with conventionalantifolates would be expected to increase the efficacy of the antifolatetreatment.

Key to the design of low molecular weight inhibitors and mechanism basedinhibitors of hGH is a knowledge of both the three dimensional structureof the active site and an identification of the catalytic mechanismincluding the catalytic amino acids. The three dimensional structure ofhGH has not been determined. However a significant advance has been madein this regard. Using sensitive sequence analysis methods to extractsubtle patterns from sequence databases, a statistical significancesimilarity was found between human gamma glutamyl hydrolase (hGH) andthe class-I glutamine amidotransferase family of enzymes. In particular,the catalytic active site from the latter is conserved in hGH as well asother amino acids near the catalytic residues.

A two step process was used to generate the sequence similarity model.First, using the sequence of hGH as the query, a search of swissprotplus trembl databases was made using the program Ssearch (Pearson 1995)which uses the Smith-Waterman algorithm to identify statisticallysignificant sequence similarities. The set of protein sequences obtainedfrom this search was then used as a starting set for the TPROBE (Neuwaldet al. 1997) program, which extracts protein alignment models fromprotein databases. Amongst the set of sequences that belong to theidentified sequence alignment model are two that have a known highresolution X-ray structure. The similarity is with the glutamineamidotransferase domain of the small subunit of carbamoyl phosphatesynthetase (CPS) from E. coli (Thoden et al. 1997), and with GMPsynthetase (Tesmer et al. 1996). In particular, the catalytic triadCys-His-Glu is strongly conserved in the sequence alignment model. Toillustrate the possible similarities, see FIG. 3 which is the proposedstructure of gamma glutamyl hydrolase (and which is the structure of thesmall subunit of CPS) showing the regions identified by the proteinalignment model. The continuous ribbon illustrates the region of humany-glutamyl hydrolase aligned to CPS.

CPS contains two domains and catalyzes the synthesis of carbamoylphosphate from bicarbonate, glutamine, and two molecules of MgATP. Thesmaller subunit is the site of glutamine hydrolysis to produce ammoniafor deliver to the larger subunit. In agreement with the site directedmutagenesis study described above, the model predicts that Cys110 in hGHis analogous to Cys 269 in CPS, which functions as the active sitenucleophile attacking the γ-carbonyl of glutamine to form a glutamylthioester intermediate (Thoden et al. 1998). The model also predictsthat His 220 and Glu 222 in hGH are the other two amino acids in thecatalytic triad, corresponding to His 353 and Glu 355 in CPS. His 220and Glu 222 are conserved in the human, rat and mouse glutamyl hydrolasesequences. In the proposed model for hGH, His 220 would activate Cys 110and Glu 222 would stabilize the resulting positively charged imidizoliumcation. Studies also show that mutating His 171 to Ala in an E. coliexpression system inactivates hGH. Interestingly the alignment modelpredicts that His 171 is analogous to His 312 in CPS. His 312 in CPSpoints away from the catalytic triad and is involved in substratebinding not catalysis. When His 312 in CPS was conservatively mutated toAsn, the K_(m) for the substrate was increased 200-fold (Miran et al.1991). A similar or greater increase in substrate K_(M) for the His 171Amutant of hGH might explain the lack of activity that was observed.Further reinforcing evidence for this alignment model was obtained byrunning the Pfam HMM search program (Sonnhammer et al. 1998). The Pfamresult has an E-value of 0.13 with the GATase model of class-I glutamineamidotransferases. Although this is not statistically significant, thealignment is similar to the one obtained using TPROBE. The alignment ofthe catalytic triad Cys-His-Glu is also found using Pfam but thisprogram does not identify the His 171 in hGH.

The proposed three-dimensional model for the active site of hGH isconsistent with the overall chemistry being catalyzed, nucleophilicattack on the gamma carbonyl of a glutamic acid amide by an activatedCys to release either glutamic acid or di gamma-Glu.

This model for the active site of hGH has been tested by preparing theCys110Ala, His220Ala, Glu222Ala, His171Ala, and His171Asn mutants ofhGH. The mutants were expressed in the baculovirus system used foractive hGH to facilitate the rapid expression and purification of theproteins. The mutant proteins were characterized for activity onmethotrexate polyglutamates. Consistent with the predictions of themodel that Cys110 and His220 are two amino acids of the catalytic triad,the mutants C110A and H220A were found to be inactive. The mutantGlu222Ala was active but had a greatly reduced activity relative to thewildtype enzyme. This is consistent with Glu222 being the third residueof the catalytic triad since when a similar mutation (Glu355Gly) wasmade in the model enzyme CPS, the mutant retained activity but it wasgreatly reduced (Hewagama et al. 1998). The role of His171 in hGH hasbeen examined by preparing the mutant His171Ala and the moreconservative mutation His171Asn. Both of these mutants were found to beinactive on methotrexate tetraglutamate suggesting that this residue inhGH plays a critical role either in binding substrate or maintaining thestructure of the enzyme.

These studies establish hGH as a cysteine peptidase of the Cys-His-Glucatalytic triad class and indicate that the catalytic site of hGH foldsin a fashion similar to CPS. The amino acids constituting the catalytictriad have been determined and His 171 is identified as a key residue inbinding substrate or maintaining the structure of the enzyme.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions and the like can bemade without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

TABLE 1 K_(m), V_(max) and specific activities of purified wildtype hGHand mutant hGH proteins V_(max) (nmol 4-NH₂-10- Specific activity (nmolCH₃PteGlu₂ 4-NH₂-10-CH₃PteGlu₂ hydrolyzed/min/μg hydrolyzed/min/mgProtein K_(m), (μM) protein) protein) Wildtype 55.2 ± 8.4  2.7 ± 0.91913 ± 32  C19A 18.1 ± 0.4^(a) 1.1 ± 0.3 1000 ± 72^(a) C124A 28.5 ±3.7^(a) 1.0 ± 0.1  725 ± 79^(a) C290A 19.5 ± 8.6^(a) 1.9 ± 0.4 1406 ±463 C110A — — no activity ^(a)compared to wildtype, p < 0.01

Results are mean+standard deviation of 3 experiments

K_(m) and V_(max) activities were measured using variable4-NH₂-10-CH₃PteGlu₂ substrate concentrations. Specific activities weremeasured using 200 μM 4-NH₂-10-CH₃PteGlu₂ for 5 minutes.

TABLE 2 Alignment of known GH amino acid sequences between amino acids16-21 and 103-118. Amino acids^(a) Species 16-21 103-118 Homo sapiens^(b) MQK C RN YFPVWGT C LGFEELSL (SEQ ID NO:11) (SEQ ID NO:12) Musmusculus ^(c) MQE C FG HFPVWGT C LGFEELSV (SEQ ID NO:13) (SEQ ID NO:14)Rattus norvegicus ^(d) MQE C YG HFPVWGT C LGLEELSV (SEQ ID NO:15) (SEQID NO:16) ^(a)amino acids highlighted in bold are conserved in all threeGH sequences; underlined are C19 and C110. ^(b)Yao et al. 1996b^(c)Esaki et al. 1998 ^(d)Yao et al. 1996a

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16 1 294 PRT Homo sapiens 1 Arg Pro His Gly Asp Thr Ala Lys Lys Pro IleIle Gly Ile Leu Met 1 5 10 15 Gln Lys Cys Arg Asn Lys Val Met Lys AsnTyr Gly Arg Tyr Tyr Ile 20 25 30 Ala Ala Ser Tyr Val Lys Tyr Leu Glu SerAla Gly Ala Arg Val Val 35 40 45 Pro Val Arg Leu Asp Leu Thr Glu Lys AspTyr Glu Ile Leu Phe Lys 50 55 60 Ser Ile Asn Gly Ile Leu Phe Pro Gly GlySer Val Asp Leu Arg Arg 65 70 75 80 Ser Asp Tyr Ala Lys Val Ala Lys IlePhe Tyr Asn Leu Ser Ile Gln 85 90 95 Ser Phe Asp Asp Gly Asp Tyr Phe ProVal Trp Gly Thr Cys Leu Gly 100 105 110 Phe Glu Glu Leu Ser Leu Leu IleSer Gly Glu Cys Leu Leu Thr Ala 115 120 125 Thr Asp Thr Val Asp Val AlaMet Pro Leu Asn Phe Thr Gly Gly Gln 130 135 140 Leu His Ser Arg Met PheGln Asn Phe Pro Thr Glu Leu Leu Leu Ser 145 150 155 160 Leu Ala Val GluPro Leu Thr Ala Asn Phe His Lys Trp Ser Leu Ser 165 170 175 Val Lys AsnPhe Thr Met Asn Glu Lys Leu Lys Lys Phe Phe Asn Val 180 185 190 Leu ThrThr Asn Thr Asp Gly Lys Ile Glu Phe Ile Ser Thr Met Glu 195 200 205 GlyTyr Lys Tyr Pro Val Tyr Gly Val Gln Trp His Pro Glu Lys Ala 210 215 220Pro Tyr Glu Trp Lys Asn Leu Asp Gly Ile Ser His Ala Pro Asn Ala 225 230235 240 Val Asn Pro Ala Phe Tyr Leu Ala Glu Phe Phe Val Asn Glu Ala Arg245 250 255 Lys Lys Asn His His Phe Lys Ser Glu Ser Glu Glu Glu Lys AlaLeu 260 265 270 Ile Tyr Gln Phe Ser Pro Ile Tyr Thr Gly Asn Ile Ser SerPhe Gln 275 280 285 Gln Cys Tyr Ile Phe Asp 290 2 885 DNA Homo sapiens 2agaccccacg gcgacaccgc caagaagccc atcatcggaa tattaatgca aaaatgccgt 60aataaagtca tgaaaaacta tggaagatac tatattgctg cgtcctatgt aaagtacttg 120gagtctgcag gtgcgagagt tgtaccagta aggctggatc ttacagagaa agactatgaa 180atacttttca aatctattaa tggaatcctt ttccctggag gaagtgttga cctcagacgc 240tcagattatg ctaaagtggc caaaatattt tataacttgt ccatacagag ttttgatgat 300ggagactatt ttcctgtgtg gggcacatgc cttggatttg aagagctttc actgctgatt 360agtggagagt gcttattaac tgccacagat actgttgacg tggcaatgcc gctgaacttc 420actggaggtc aattgcacag cagaatgttc cagaattttc ctactgagtt gttgctgtca 480ttagcagtag aacctctgac tgccaatttc cataagtgga gcctctccgt gaagaatttt 540acaatgaatg aaaagttaaa gaagtttttc aatgtcttaa ctacaaatac agatggcaag 600attgagttta tttcaacaat ggaaggatat aagtatccag tatatggtgt ccagtggcat 660ccagagaaag caccttatga gtggaagaat ttggatggca tttcccatgc acctaatgct 720gtgaaccccg cattttattt agcagagttt tttgttaatg aagctcggaa aaagaaccat 780cattttaaat ctgaatctga agaggagaaa gcattgattt atcagttcag tccaatttat 840actggaaata tttcttcatt tcagcaatgt tacatatttg attga 885 3 43 DNA Homosapiens 3 gcccatcatc ggaatattaa tgcaaaaagc ccgtaataaa gtc 43 4 43 DNAHomo sapiens 4 gactttatta cgggcttttt gcattaatat tccgatgatg ggc 43 5 34DNA Homo sapiens 5 cctgtgtggg gcacagcgct tggatttgaa gagc 34 6 34 DNAHomo sapiens 6 gctcttcaaa tccaagcgct gtgccccaca cagg 34 7 35 DNA Homosapiens 7 gctgattagt ggagaggcct tattaactgc cacag 35 8 35 DNA Homosapiens 8 ctgtggcagt taataaggcc tctccactaa tcagc 35 9 37 DNA Homosapiens 9 cttcatttca gcaagcttac atatttgatt gaaagtc 37 10 37 DNA Homosapiens 10 gactttcaat caaatatgta agcttgctga aatgaag 37 11 6 PRT Homosapiens 11 Met Gln Lys Cys Arg Asn 1 5 12 16 PRT Homo sapiens 12 Tyr PhePro Val Trp Gly Thr Cys Leu Gly Phe Glu Glu Leu Ser Leu 1 5 10 15 13 6PRT Mus musculus 13 Met Gln Glu Cys Phe Gly 1 5 14 16 PRT Mus musculus14 His Phe Pro Val Trp Gly Thr Cys Leu Gly Phe Glu Glu Leu Ser Val 1 510 15 15 6 PRT Rattus norvegicus 15 Met Gln Glu Cys Tyr Gly 1 5 16 16PRT Rattus norvegicus 16 His Phe Pro Val Trp Gly Thr Cys Leu Gly Leu GluGlu Leu Ser Val 1 5 10 15

What is claimed is:
 1. A molecule capable of binding to one or more ofamino acid residues 110, 171, 220 or 222 in the amino acid sequence ofgamma glutamyl hydrolase as shown in SEQ ID NO:1, wherein the moleculemodifies the activity of gamma glutamyl hydrolase and wherein themolecule has a three dimensional structure complementary to the threedimensional structure of gamma glutamyl hydrolase in a fragment thatincludes one or more of the one or more of amino acid residues 110, 171,220 or
 222. 2. The molecule of claim 1 wherein the molecule is anantibody.
 3. The molecule of claim 1 wherein the molecule is a peptide.4. A composition comprising the molecule of claim 1 and a suitablecarrier.
 5. A composition comprising the molecule of claim 1 and anantifolate.
 6. A method of increasing the effectiveness of antifolatetreatment, the method comprising co-administering the molecule of claim1 with an antifolate.
 7. A method of modifying the activity of gammaglutamyl hydrolase protein, the method comprising: providing a gammaglutamyl hydrolase protein; and exposing the gamma glutamyl hydrolaseprotein to the molecule of claim 6, wherein the molecule binds to one ormore of amino acid residues 110, 171, 220 or 222 in the amino acidsequence of the%gamma glutamyl hydrolase protein thereby modifying theactivity the gamma glutamyl hydrolase protein.
 8. A method ofidentifying a molecule that modifies the activity of gamma glutamylhydrolase protein, the method comprising: determining whether a moleculebinds to one or more of amino acid residues 110, 171, 220 or 222 in theamino acid sequence of gamma glutamyl hydrolase as shown in SEQ ID NO:1;and screening a molecule that binds to one or more of amino acidresidues 110, 171, 220 or 222 to determine whether the screened moleculemodifies the activity of gamma glutamyl hydrolase protein.
 9. A moleculeidentified by the method of claim
 8. 10. A method of modifying theactivity of gamma glutamyl hydrolase protein, the method comprising:providing a gamma glutamyl hydrolase protein; and exposing the gammaglutamyl hydrolase protein to the molecule of claim 9, wherein themolecule binds to one or more of amino acid residues 110, 171, 220 or222 in the amino acid sequence of the gamma glutamyl hydrolase proteinthereby modifying the activity of the gamma glutamyl hydrolase protein.